Method of treating certain tumors using illudin analogs

ABSTRACT

A therapeutic method is provided to inhibit tumor cell growth in a subject in need of said therapy is provided, comprising parenterally administering a therapeutic amount of a compound in a pharmaceutically acceptable carrier, wherein said compound has the structure: ##STR1## wherein R is selected from the group consisting of: ##STR2## and wherein said tumor cell is sensitive to inhibition by said compound and is selected from the group consisting of a myeloid leukemia cell, a T-cell leukemia cell, a lung carcinoma cell, an ovarian carcinoma cell, and a breast carcinoma cell.

This work was supported in part by research grant CA-37641 from theNational Institutes of Health. The U.S. Government has rights in theinvention.

This application is a continuation-in-part of U.S. Ser. No. 07/606,511,filed Oct. 31, 1990; which is a continuation-in-part of U.S. Ser. No.07/416,395, filed Oct. 3, 1989, now abandoned the content of which ishereby incorporated by reference into the present application.

BACKGROUND OF THE INVENTION

Multiple agent chemotherapy has curative potential in some hematologicmalignancies and advanced rapidly proliferating solid tumors. Curativechemotherapy has benefitted from the discovery of new, relativelynon-cross resistant agents, and more effective use of existing agents.Interventions which increase the efficacy of conventional agents includemore effective regimens for multiple drug administration, minimizationof drug toxicities and increased use of adjuvant, surgical or radiationtherapy.

Despite recent advances, patients with many types of malignancies remainat significant risk for relapse and mortality. After relapse, somepatients can be reinduced into remission with their initial treatmentregimen. However, higher doses of the initial chemotherapeutic agent orthe use of additional agents are frequently required, indicating thedevelopment of at least partial drug resistance. Recent evidenceindicates drug resistance can develop simultaneously to several agents,including ones to which the patient was not exposed. The development ofmultiple-drug resistant (mdr) tumors may be a function of tumor mass andconstitutes a major cause of treatment failure. To overcome this drugresistance, high-dose chemotherapy with or without autologous bonemarrow transplantation is employed. The high-dose chemotherapy mayemploy the original drug(s) or be altered to include additional agents.The feasibility of this approach has been demonstrated for hematopoieticand solid tumors. The development of new drugs non-cross resistant withmdr phenotypes is required to further the curative potential of currentregimens and to facilitate curative interventions in previously treatedpatients.

Recently, the in vitro anti-tumor activity of a novel class of naturalproducts called illudins was examined in Kelner, M. et al., Cancer Res.47:3186 (1987), incorporated herein by reference. Illudin S and M aretwo types of illudins known to exist. Illudins have a chemical structureentirely different from other chemotherapeutic agents. Illudin compoundswere previously purified and submitted for evaluation to the NationalCancer Institute Division of Cancer Treatment (NCI DCT) in vivo drugscreening program but had a low therapeutic index in other experimentaltumor systems in accordance with NCI studies. The extreme toxicity ofilludins has prevented any applications in human tumor therapy.

Thus, these exists a need for chemotherapeutic agents which are toxic totumors, and especially mdr tumors, and have an adequate therapeuticindex to be effective for in vivo treatment. The subject inventionsatisfies this need and provides related advantages.

SUMMARY OF THE INVENTION

A therapeutic method of inhibiting tumor cell growth in a subject inneed of said therapy is provided, comprising parenterally administeringa therapeutic amount of a compound in a pharmaceutically acceptablecarrier, wherein said compound has the structure: ##STR3## wherein R isselected from the group consisting of: ##STR4## and wherein said tumorcell is sensitive, to inhibition by said compound and is selected fromthe group consisting of a myeloid leukemia cell, a T-cell leukemia cell,a lung carcinoma cell, an ovarian carcinoma cell, and a breast carcinomacell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the sensitivity of breast carcinoma and myeloid leukemiacells versus other tumors to illudin S.

FIG. 2 shows the active sites of illudin S.

FIG. 3 shows an NMR spectrum demonstrating the presence of a shortacting intermediate in acid. Signal A is from the hydrogen on the doublebond in the 5 membered ring (illudin M). Signal B is from the hydrogenatom on the short lived intermediate that results from the cyclopropanering opening up (but before the double bond reacts). Signals marked at Care from the product that results when the double bond has reacted. Withtime, the signal peaks from illudin M will disappear and the peaks atposition C will be the predominate signals. Signal B will disappearconcurrently with Signal A confirming it is a short lived intermediatearising from illudin M.

FIG. 4 shows the effect of illudin S on Molt-4 tumor growth in athymicmice (Balb/c).

FIG. 5 shows the effect of dehydroilludin M on tumor growth.

FIG. 6 shows the response of HL60/MRI xenograft to dehydroilludin M.

FIG. 7 shows illudin S uptake using relatively sensitive HL60 cells andresistant B cells.

FIG. 8 shows the rapid intracellular accumulation of illudin S by HL60cells was saturated at high concentrations.

FIG. 9 shows the analysis of the initial uptake of illudin S by HL60cells at varying concentrations conformed to Michaelis-Menton saturationconstants.

FIG. 10 shows the effect of 6-hydroxymethylacylfulvene and otherconventional anticancer agents on human lung adenocarcinoma MV522.

FIG. 11 shows the effect of different doses of6-hydroxymethylacylfulvene on human lung adenocarcinoma MV522.

DETAILED DESCRIPTION OF THE INVENTION

A method of inhibiting tumor cell growth in a subject is providedcomprising contacting the tumor with a therapeutic amount of an illudinS or illudin M analog having the structure: ##STR5## wherein R is##STR6##

By "inhibiting" is meant either decreasing the tumor cell growth ratefrom the rate which would occur without treatment or causing the tumorcell mass to decrease in size. Inhibiting also includes a completeregression of the tumor. Thus, the analogs can either be cytostatic orcytotoxic to the tumor cells enumerated above.

The subject can be any animal having a myeloid leukemia, a T-cellleukemia, a lung carcinoma, an ovarian carcinoma or breast carcinoma.The analogs are effective on human tumors in vivo as well as on humantumor cell lines in vitro.

The tumors listed hereinabove can be contacted with the analog by anyeffective means, many of which are well known in the art. The route ofadministration to the subject can include intravenous, oral,intraperitoneal, and oral and nasal inhalation. The preferred route ofadministration depends on the subject and type of tumor encountered.

Applicants have made the surprising discovery that analogs of illudin Sand M can be made which are less toxic than illudin S and M but are amore effective chemotherapeutic agent in vivo. As noted above, illudin Sand M have a low therapeutic index due to the extreme toxicity and,therefore, cannot be used therapeutically in humans. Applicants havediscovered that various modifications in illudin S and M inhibitnucleophiles from reacting with the compound. This results in lessfacile opening of the cyclopropane ring and reduces the toxicity of thecompound in vivo while resulting in a high therapeutic index. TheApplicants have made the further discovery that an acylfulvene analog,6-hydroxymethylacylfulvene, is markedly more efficacious and nontoxicthan previously known S and M illudin analogs.

The therapeutically effective amount of analog varies with the subject.However, it has been found that relatively high doses of the analogs canbe administered due to the decreased toxicity compared to illudin S andM. A therapeutic amount between 30 to 112,000 μg per kg of body weighthas been found especially effective for intravenous administration while300 to 112,000 μg per kg of body weight is effective if administeredintraperitoneally. As one skilled in the art would recognize, the amountcan vary depending on the method of administration. Further, the amountcan vary if the analog is linked to a toxin. The methods of theinvention are especially effective against tumor cells of myeloid,epidermoid, T-cell leukemia, and lung, ovarian and breast carcinoma.

EXAMPLE I Synthesis of Dehydroilludin M

A mixture of illudin M (200 mg) and pyridinium dichromate (1 g) in drydichloromethane (60 ml) was stirred at room temperature in a flaskequipped with a rubber septum so that an atmosphere of argon could bemaintained. After 20 hours, the reaction mixture was diluted withdiethyl ether (20 ml) and filtered through a short column of silica gel.The column was further eluted with more diethyl ether and the combinedfiltrate was concentrated, giving a residue which was chromatographed onsilica gel with hexane-ethyl acetate (10:1) as eluent. The desiredcompound was obtained in early fractions from the chromatography. Theyield was 140 mg of white crystals melting at 64°-65° C. NMR spectraldata were recorded for this compound.

EXAMPLE II Synthesis of Fulvene

Illudin S (50 mg) was dissolved in water (2 mL) and 3N hydrochloric acid(2 mL) added to the solution. The resulting solution soon became cloudy(within 30 min) and a yellow precipitate formed. The mixture was placedin the refrigerator overnight; then it was extracted with chloroform (10mL). The yellow chloroform solution was dried (MgSO₄) and the solventwas removed under reduced pressure leaving an orange-yellow gum. Thismaterial was chromatographed on silica gel with hexanes: ethyl acetate(6:1) as eluent giving the fulvene (20 mg) and the bisfulvene (10 mg).NMR spectral data were recorded for these compounds.

Alternatively, a total synthesis of the fulvene can also be achieved inthe following way: ##STR7##

Reaction of the known 1, 1-diacetyl cyclopropane with the dianion of thecyclopentadiene derivative shown gives a diol which on mild acidtreatment gives the diolketone. Selective elimination of a tertiaryhydroxyl group gives the desired fulvene.

EXAMPLE III In Vitro Studies

To assess cytotoxic effects, various concentrations of illudins wereadded to cultures of cells for 48 hours, then cell growth/viability wasdetermined by trypan blue exclusion. As an alternative to 48 hourcontinuous exposure studies, cells were plated in liquid culture in 96well plates, exposed to various concentrations of illudins for 2 hours,pulsed with [³ H]-thymidine for one to two hours and harvested ontoglass filters. The filter papers were added to vials containingscintillation fluid and residual radioactivity determined in a beta(scintillation) counter.

When screening the sensitivity of other solid tumor cell lines toilludin S, a breast cell line, MCF-7, was noted to be markedly sensitive(FIG. 1). Another breast cell line maintained in our laboratory,MDA-231, was also found to be markedly sensitive to illudin S (FIG. 1).

Studies with dehydroilludin M indicated this analog also displayedselective toxicity towards myeloid leukemia cells and breast carcinomalines MCF-7 and MDA-231 (Table 1).

                  TABLE 1                                                         ______________________________________                                        Histiospecific cytotoxicity of illudin S and dehydroilludin                   M as demonstrated by inhibition of thymidine after a two                      hour exposure to the toxins (N - 3).                                                                    IC.sub.50 (nM/L)                                    Compound        Illudin S Dehydroilludin M                                    ______________________________________                                        HL60, myeloid   7 ± 1  246 ± 19                                         8392, B-cell    236 ± 31                                                                             >38,000                                             8402, T-cell    669 ± 196                                                                            >38,000                                             242, melanoma   607 ± 70                                                                             >38,000                                             547, ovarian    607 ± 110                                                                            >38,000                                             SL-2, murine (thymic)                                                                         142 ± 15                                                                             5,235 ± 277                                      MCF-7, breast   58 ± 5 653 ± 65                                         MDA-231, breast 2.0 ± 0.2                                                                            112 ± 17                                         ______________________________________                                    

Because previous studies showed that CEM mdr variants were not resistantto illudin S, several other mdr cell types were studied forsusceptibility to illudin S and the dehydroilludin M. These mdr daughtercell lines demonstrate a 200 to 800 fold increase in resistance tomultiple conventional chemotherapeutic agents, but showed minimal or noresistance to illudin S or dehydroilludin M (Table 2). Thus, mdr cellsassociated with or without the gp170 protein were still susceptible toilludin toxicity. These studies indicate that illudins' novel structureconfers relative non-cross resistance in multidrug resistanthematopoietic cell lines. The derivative of illudins, dehydroilludin M,is slightly less toxic than the parent illudin compound, but results(Table 2) indicate that there is no cross-resistance to this compound invarious mdr cell lines.

The effect of illudin S and dehydroilludin M on L1210, murine bonemarrow CFU-gm, and C1498 (AML cell line) was studied. Illudin S was themost potent agent ever tested in this assay and displayed the largestdifferential effect ever noted between L1210 and AML leukemia lines andCFU-gm zone cites (Table 3). The derivative, dehydroilludin M, whileless toxic was markedly more selective towards the AML line. Itinhibited AML colony formation at concentrations where it had no effecton the CFU-gm cells (Table 4).

                  TABLE 2                                                         ______________________________________                                        Sensitivity of Different Mdr Lines to Illudin S                                                            Dehydroilludin                                   MDR cell line available                                                                          Illudin S M                                                ______________________________________                                        CEM Variants                                                                            Parent       8.3 ± 2.6                                                                            nt                                                     VM-1         16.2 ± 6.4                                                                           nt                                                     AraC         14        nt                                                     VLB100       3.7 ± 0.7                                                                            nt                                                     (gp170+)                                                                      Dox (gp170+) 14                                                     MDA-231   Parent       0.85 ± 0.23                                                                          54 ± 7                                    (Breast)  3-1 (gp170+) 0.89 ± 0.38                                                                          58 ± 11                                   MCF7-wt   Parent       0.88 ± 0.11                                                                          92 ± 15                                   (Breast)  ADR (GSH-    3.7 ± 0.4                                                                            68 ± 15                                             transferase)                                                        HL-60     Parent       3.1 ± 1.1                                                                            163 ± 11                                            ADR (gp150+) 1.9 ± 0.8                                                                            191 ± 44                                  KB variant                                                                              Parent       0.58 ± 0.12                                                                          125 ± 14                                            C-1 (gp170+) 0.69 ± 0.15                                                                          80 ± 18                                             VBL (gp170+) 0.69 ± 0.11                                                                          78 ± 19                                   L1210     Parent       0.42 ± 0.08                                                                          62 ±  8                                             DDPt (cis-plat)                                                                            0.46 ± 0.12                                                                          119 ± 39                                            BCNU         0.58 ± 0.08                                                                          100 ± 31                                            PAM (melphalan)                                                                            0.62 ± 0.15                                                                          73 ± 31                                             CPA (cyclophos)                                                                            0.46 ± 0.12                                                                          38 ± 15                                   ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Inhibition of Growth by Illudin S                                             Illudin S Concentration                                                                       Zone of Inhibition                                            (ug/disc)       L1210      Go     Colon 38                                    ______________________________________                                        2.50            500        240    30                                          1.25            400        70     0                                           0.63            320        30     0                                           ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Effect of Illudins on Colony Formation                                                     Zone Size                                                        Compound  Dilution L1210   CFU-GM  C1498 (AML)                                ______________________________________                                        Illudin S 1/1,000  850     400     >1000                                                1/4,000  600     200     800                                                  1/16,000 550      0      550                                                  1/64,000 300      0      250                                        Dehydroilludin                                                                          1/25     400     200     >1000                                      M         1/125    200     100     750                                                  1/125    300      50     700                                                  (repeat)                                                                      1/625    100      0      400                                        ______________________________________                                    

EXAMPLE IV Structure Function Studies

The structure-function studies were performed by synthesizingderivatives of the illudins and examining their in vitro toxicity forHL60 leukemia cells (Table 5). This study identified three criticalsites for illudin toxicity. These include the cyclopropane ring (siteA), the alpha/beta unsaturated bond site (site B), and the ketone group(site C) (FIG. 2). Alteration of any of these sites resulted in up to a4 log decrease in toxicity. In contrast, the non-ring primary hydroxylgroup (FIG. 2, site D) does not contribute to toxicity. Various largechemical groups can be attached to this site without altering toxicity.Many of the derivatives with a marked decrease in toxicity (as comparedto illudin S or M) are still more potent than conventionalchemotherapeutic agents such as BCNU or cis-platinum (Table 5).

                  TABLE 5                                                         ______________________________________                                        IC.sub.50 for Various Illudin Derivatives Versus                              Other Agents in HL-60 cells                                                   COMPOUNDS              nM                                                     ______________________________________                                        Illudin S or M         10                                                     Dihydroilludin S or M  100,000                                                Acylfulvene            500                                                    Dehydroilludin M (diketone)                                                                          246                                                    Isoilludin M           3,800                                                  Ptaquiloside           7,700                                                  Pterosin C             12,500                                                 2,5,6,7-tetramethylindenone                                                                          475                                                    Illudin tosylate       38                                                     DNA polymerase inhibitor: Aphidocolin                                                                2,100                                                  Alkylating agent: BCNU 23,300                                                 Crosslinking agent: cis-platinum                                                                     550 ± 14                                            Alkylating agent: MNNG 15,000                                                 Protein Synthesis Inhibitor: Ricin                                                                   0.2                                                    ______________________________________                                    

EXAMPLE V Structure-Function Studies: Chemical

Illudin M is readily converted to stable aromatic compounds (ontreatment with dilute HCl) which in cell culture studies are more than1,000 fold less toxic. The chlorine-carbon bond formation, cyclopropanering opening and extrusion of the tertiary hydroxyl (as water) aresynchronous. The intermediate formed can be detected by NMR spectroscopyof the reaction mixture (FIG. 3). The intermediate, however, is highlyreactive and is quickly converted to a phenol by attack of a secondnucleophile, i.e., water. Thus, under acidic conditions, illudin M isclearly bifunctional.

The above studies indicate that the toxicity of illudins is related tothe ease with which the tertiary hydroxyl can be removed and thecyclopropane ring opened. It was found that illudin toxicity depends onthe combined effects of the cyclopropane group (site A, FIG. 2), the twodouble bonds (conjugated diene) (site B), and the ketone (site C). Itwas hypothesized that oxidation of the secondary hydroxyl group in thefive membered ring to a ketone would alter the potency or selectivity ofthe molecule by contributing to further electron delocalization withinthe molecule. The new ketone group acts as an "electron sink" so thatelectrons of the cyclopropane C-C bonds are delocalized towards theketone rather than to the carbon atom bearing the tertiary hydroxyl.This means the incipient carbocation, forming as the carbon-oxygen(oxygen of the tertiary hydroxyl) bond breaks, is not as stable as inthe case of illudin M. Therefore, carbon-oxygen bond breaking is lessfavorable and reactivity is reduced. This ketone derivative, termeddehydroilludin M, was synthesized and was less toxic to HL-60 cells invitro than illudin S or M (Table 4). As discussed above, the toxicity ofdehydroilludin M appeared relatively selective for myeloid and breastcarcinoma cells in vitro (FIG. 1 and Table 1).

Consistent with the above hypothesis are the results of the kinetics ofthe reaction of illudin M and dehydroilludin M with dilute HCl. Indilute HCl, illudin M undergoes a pseudo first-order reaction(k=4.7×10⁻³ min⁻¹, tl/2=148 minutes) Dehydroilludin M also demonstratedfirst-order kinetics but the reaction was considerably slower (k=2×10⁻⁴min¹ tl/2=2765 min). In the reaction with dehydroilludin M, nointermediate could be detected by NMR spectroscopy. Presumably it formedtoo slowly and is too short-lived to be detected. The lower reactivityshown by dehydroilludin M suggests it is more selective in its reactionwith nucleophiles and thus has a lower toxicity compared to illudin M.

The reaction of illudins with a naturally occurring nucleophile,glutathione has also been studied. At a wide pH range, from pH 3 to pH9, glutathione spontaneously reacts with illudin M, illudin S, ordehydoilludin M, producing products analogous to those from the reactionof illudin M and HCl. The reaction rate is optimized at a pH of 6.1 to7.0, indicating the reaction could occur intracellularly.

The toxicity of illudins towards a breast cell carcinoma line MCF7-wtand its MDR resistant daughter line MCF/Adr was then studied. The gp170negative daughter cell line is drug resistant on the basis of a 50 foldincrease in glutathione transferase, which results in a 200 to 800 folddecrease in sensitivity to conventional chemotherapeutic agents. Thisline also shows a 4.1 fold decrease in glutathione content. Thisdaughter line showed a 4.2 fold decrease in sensitivity to illudin S(parent IC₅₀ 0.88 nmoles/1; daughter line 3.70 nanomoles/1) versus the200 to 800 fold seen with other agents. Kinetic studies on the abilityof illudins to inhibit glutathione transferase indicated there was nodirect inhibition of enzyme activity. These findings show that whileilludin toxicity is inversely correlated with intracellular glutathionecontent it is not correlated with glutathione transferase activity.

EXAMPLE VI Animal Studies

Using procedures set forth in Leonard, J. E. et al., Cancer Res.47:2899-02 (1987) and Dillman, R. O. et al, Cancer Res. 45:5632-36(1985), both incorporated by reference herein, Molt-4 (human T-cellleukemia) xenografts were established in four week old athymic Balb/cnu/nu mice. After 3 weekly doses of total body radiation (600 cGy), micewere given subcutaneous flank injections of Molt-4 cells together withirradiated (6000 cGy) HT-1080 feeder cells. Two animals received onlyirradiated HT-1080 feeder cells to ensure these cells did not inducetumors. Animals were monitored for Molt-4 tumor development and whentumors were palpable (approximately 4×4 mm at 5 to 7 days), mice wererandomized into groups of 5 as previously described. Control micereceived intraperitoneal saline and treated mice received either 300μg/kg illudin S, 30 or 300 μg/kg dehydroilludin M, IP twice weekly. Inmice given illudin S there was tumor growth delay (FIG. 4).

In contrast, in nude mice which received the dehydroilludin M at the lowdosage of 30 μg/kg (the compound was subsequently found to be nontoxicto mice at 60,000 μg/kg IP twice a week), three of five tumors underwentcomplete regression, but two tumors failed to respond (FIG. 5). The twoapparently resistant tumors were harvested and tested in vitro forresistance to illudin S and dehydroilludin M. There was no evidence ofresistance to either compound. Two of the complete responders werefollowed for over twelve weeks without evidence of tumor regression.

Using a different source of athymic nude mice, these experiments wererepeated. In these studies there was little effect of illudins on tumorgrowth. The reason for this variability in response to Molt-4 xenograftsprobably relates to the low doses of dehydroilludin M, interanimalvariations in glutathione metabolism, or drug distribution.

The efficacy of dehydroilludin M was then screened in a syngeneic modelusing murine SL-2 cells. SL-2 leukemia/lymphoma cells are injectedsubcutaneously and metastasized to lymph nodes, spleen, and lungs, anddrug efficacy in this model is determined by increased life span (ILS).The SL-2 cells were administered at 2.5 million cells per animal andtreatment was delayed for 7 days until the tumors were palpable. This isa relatively stringent test against established tumors and contrasts togeneral drug screens in the SL-2 model which normally use only 0.5million cells and starting drug treatment at 3 days. Dehydroilludin Mhad a little effect at 30 mg/kg IP twice a week, ILS 5%, and 60 mg/kg IPtwice a week, ILS 18%. When administered IV at 0.03 mg/kg, twice a week,the ILS increased to 38%. This suggests the drug is metabolized by theliver and is likely more efficacious when administered IV.

During the course of these in vivo experiments, it became clear from invitro experiments, that histiospecificity of illudins depends upon thepresence of an active energy-dependent pump. The SL-2 and the Molt-4cells were studied and it was determined that the uptake mechanism wasnot present. Therefore, the studies were redirected into xenograftmodels that used cells of myeloid lineage.

Human HL-60 cells capable of growing as xenografts in nude mice withoutanimal radiation were obtained from Dr. Theodore Brightman (NCI). Thesecells termed HL-60 MRI cells, were confirmed to have energy-dependentuptake pump, a not unexpected finding as their parental cells possessthe pump. Dehydroilludin M induced dose related tumor inhibition whenadministered IP on a twice a week schedule (FIG. 6). The MTD IP dose fordehydroilludin M was reached in these studies on the 2 dosages per weekIP dose schedule. Similar tumor regressions have been observed with IVdehydroilludin M.

In collaboration, the in vivo effects of dehydroilludin M was againstudied. Initially the compound was studied against L1210 cells. A doseof 2.5 mg/kg IP given daily for 5 days resulted in an ILS of only 9%.The dehydroilludin M was then administered as a 24 hour infusion (5.0mg/kg); the ILS was 11%. After we became aware of the presence of theenergy-dependent uptake in human myelocytic cells, dehydroilludin M wasscreened for in vivo efficacy against a syngeneic mouse AML model usingC1498 cells and a single bolus of illudin S, 2.5 mg/kg IP, produced anILS of 35%. A second trial using the same dosage, administered IP once aday for 5 days resulted in a 44% ILS. As the animals can tolerate 60mg/kg IP or 1 mg/kg IV (tail vein) on a twice a week schedule for 4weeks without demonstrating weight loss or a decrease in food/waterintake, it is possible to further optimize both dosage and treatmentschedule.

EXAMPLE VII HL60/MRI Mouse Experiment With Acylfulvene andDehydroilludin M

Thirty mice were injected subcutaneously, over the shoulder, with500,000 HL60/MRI cells (human myeloid leukemia tumor cells). Treatmentwas begun on day 11, rather than immediately. This delay in startingtreatment is a stringent test; to determine whether a compound iseffective. By delaying treatment, the tumor cells become firmlyestablished.

The mice were divided into 6 groups of 5 each. One group was the controland these animals received on a placebo, the solution used to dilute theagent. The other groups received the following compounds and dosages:the dehydroilludin M compound at 1.0 mg/kg, the dehydroilludin M at 3.0mg/kg, the Acylfulvene at 0.3 mg/kg, the Acylfulvene at 1.0 mg/kg, theAcylfulvene at 3.0 mg/kg. All animals received the placebo or drugs byintravenous injection using a tail vein. The placebo or drugs wereadministered on a twice a week schedule.

Results are summarized in the accompanying table 6. Both thedehydroilludin M and the Acylfulvene compound were effective atinhibiting tumor growth and demonstrated dosage dependence inhibition(the more drug administered, the less the tumors grew). The animalsreceiving the highest amount of either drug did not display any evidenceof adverse effect, such as a decrease in food or water intake, nor astatistically significant decrease in body weight. These results showthat higher dosages of either drug can be administered. Also, that thedrug could be administered on a more effective dosage schedule, such ason a daily basis.

                                      TABLE 6                                     __________________________________________________________________________    Summary: HL60/MRI experiment, intravenous - #1                                BY TOTAL TUMOR WEIGHT [Mg]                                                    DAY 11      DAY 18                                                                              DAY 25 DAY 32  DAY 40                                       __________________________________________________________________________    CONTROL                                                                       No Drug                                                                             99 ± 36                                                                          845 ± 282                                                                        3299 ± 1080                                                                       10162 ± 4123                                                                       16747 ± 5061                              Dehydroilludin M                                                              1 mg/kg                                                                             114 ± 55                                                                         883 ± 311                                                                        2274 ± 992                                                                        6025 ± 1772                                                                        11507 ± 3707                              IV                                                                            3 mg/kg                                                                             101 ± 40                                                                         911 ± 309                                                                        2127 ± 1092                                                                       2854 ± 1260                                                                        4784 ± 2303                               IV                                                                            Acylfulvene                                                                   0.3 mg/kg                                                                           73 ± 38                                                                          540 ± 167                                                                        1352 ± 520                                                                        3204 ± 1147                                                                        9501 ± 4605                               IV                                                                            1 mg/kg                                                                             58 ± 32                                                                          582 ± 297                                                                        964 ± 685                                                                         2321 ± 1434                                                                        6275 ± 2865                               3 mg/kg                                                                             38 ± 30                                                                          369 ± 250                                                                        336 ± 215                                                                         437 ± 238                                                                          1201 ± 501                                __________________________________________________________________________

EXAMPLE VIII General In Vitro Screening Procedures and Cell UptakeStudies

In keeping with the suggestions of the previous examples and ourconcentration on mechanisms of illudin action and tissue specificity,other myeloid leukemia cell lines can be screened for rapid illudinuptake (KGl, KGla, HEL, K562, OCI-Ml, AML-193).

The procedures for in vitro screening of illudin compounds are detailedin the previous examples. Cytotoxicity of new analogs for cell lines isinitially evaluated over a 5 log range using growth or semi-solid colonyforming assays, and inhibition of thymidine incorporation. Inhibition ofthymidine incorporation is used because earlier studies indicate thatthymidine incorporation is preferentially inhibited by illudins andcorrelates closely with cell death. Analogs are screened against normalbone marrow progenitors and a variety of cell lines involving variousleukemias, B and T cell) and solid tumors (melanoima, ovarian).

In vitro testing of dehydroilludin M on various cell lines, includingMDR lines, can also be performed on DNA-repair deficient cell lines andnormal bone marrow progenitors. A variety of other analogs can beprepared. Since these analogs will have alterations in the known activesites, they are expected to result in a similar tumor inhibition.Screening studies for these analogs can include various mdr cells (toensure that no cross-resistance occurs) and DNA-repair deficient celllines.

In vitro testing can also study sensitivity of other breast cell linesto determine if they are also preferentially sensitive to illudin S,dehydroilludin M, and the fulvene analog.

EXAMPLE IX Assessment of Illudin Uptake in Tumor Cells

While human myeloid tumor cells are sensitive to illudins, their normalprecursors, granulocyte/macrophage forming units, are relativelyresistant to illudins by 1.5 to 2.0 logs, demonstrating that thetransport system is absent from some normal marrow cells and providing atherapeutic margin of safety.

Specific illudin S uptake was assayed using relatively sensitive HL60cells and resistant B cells. At 37° C., HL60 myeloid leukemia cellsdemonstrated rapid uptake of illudin S, while the relatively insensitive8392 B-cells exhibited comparatively little drug incorporation (FIG. 7).The intracellular accumulation of illudins in the B cell line was slowand linear for 7 hours (r-0.984), at which time the intracellularconcentration approached that of the incubation mixture. HL60 cells, incontrast, rapidly accumulated the toxin and intracellular accumulationreach a plateau within one hour. HL60 cells exposed to 10 nM illudin Sconcentrated the toxin 19 fold, whereas B cells did not activelyconcentrate the toxin. The rapid intracellular accumulation of illudin Sby HL60 cells was saturated at high concentrations (FIG. 8). Incontrast, illudin S accumulation in 8392 B cells remained concentrationdependent. Analysis of the initial uptake of illudin S by HL60 cells atvarying concentrations revealed that the influx of illudin S conformedto Michaelis-Menton saturation kinetics (FIG. 9). The Vmax for HL 60cells was 27 picomoles/minute/mg of protein and the Km was 4.2 μM. Thisindicates HL60 cells have a very high transport capacity for illudins asthe Vmax for illudins is 5 times the Vmax for folate, a vitamin requiredby cells.

Cold (4° C.), 1% azide, and the metabolic blockers 2-deoxyglucose andantimycin A, all block uptake of illudin S into HL60 cells but havelittle effect on the insensitive 92 B-cells (Table 7). These studiesindicate that illudin S is transported and concentrated into HL60 cellsby an energy dependent transport system, whereas the transport intoinsensitive B-cells occurs only by diffusion (passive or nonenergyrequiring transport). MCF7 breast tumor cells also demonstratedinhibition of uptake by cold. The finding of an energy-dependenttransport mechanism explains why myeloid and breast tumor cells are sosensitive to illudins with short exposure times, but B-cells are not.

                  TABLE 7                                                         ______________________________________                                        Uptake of [.sup.3 H] Illudin S by HL60 Myeloid versus                         8392 B-cells                                                                  Maximum uptake per hour (picomoles).sup.a                                     Conditions   HL60        8392     MCF7                                        ______________________________________                                        37° C.                                                                              .sup. 75 ± 16.sup.b                                                                    5.5 ± 1.4                                                                           29 ± 4                                    4° C.                                                                              4.3 ± 0.9                                                                              3.4 ± 1.0                                                                           4.0 ± 2.1                                1% Azide     8.7 ± 1.4                                                                              4.3 ± 1.3                                                                           NT.sup.b                                    2-deoxyglucose &                                                                           16.7 ± 3.5                                                                             3.6 ± 1.4                                                                           NT.sup.                                     Antimycin A                                                                   ______________________________________                                         .sup.a per 10 million cells                                                   .sup.b NT = not tested                                                   

Cells were exposed to 100 ng/ml of [³ H]-labeled illudin S for one hourand harvested as described. Results are expressed as mean ±SE andrepresent 3 experiments.

EXAMPLE X Synthesis and Structure of 2,5,6,7-Tetramethyl-1-Indenone andDehydropterosin Compounds

First 2,4,5,6-tetramethyl-1,3-indanione was synthesized by preparing asolution of 1,2,3-trimethylbenzene and methylmalonylchloride in carbondisulfide and adding aluminum trichloride dropwise over two hours. Themixture was relfuxed for 2 more hours, crushed ice added, and extractedthree times with chloroform. The combined extract was washed with brine,dried, and solvent removed to leave a residue which was purified bychromatography with 1% ethyl acetate in benzene. Removal of solvent andpurification by sublimation gave the desired product.

The 2,5,6,7-tetramethyl-1-indenone was prepared by reducing2,4,5,6-tetramethyl-1,3-indanione with zinc dust at 50° C. Product waspurified by chromatography with 1% ethyl acetate in benzene to yield twoisomers. The major isomer was treated with 10% potassium hydroxide, thenpurified by sublimation. The compound has the structure: ##STR8##Dehydropterosin O synthesis: 3-acetoxy-6(beta-methoxy)ethyl-2,5,7-trimethyl-1-indanone was dissolved in tetrahydrofuran and10% potassium hydroxide and refluxed for two hours. The solution wasthen extracted three times with ether and the combined extractschromatographed with 2% ethylacetate in benzene to yield theDehydropterosin O compound. The compound has the structure: ##STR9## R=HDehydropterosin B R=CH₃ Dehydropterosin O

Both compounds were toxic to cells in vitro and have antifungalproperties.

EXAMPLE XI Synthesis of Acylfulvene Analogs

Many acylfulvene analogs possessing the key structural features requiredfor antitumor activity may be prepared starting from illudin S or bytotal synthesis from simple precursors. Illudin S is produced byfermentation of Omphalotus illudens. On dissolving this compound inwater and adding dilute H₂ SO₄ it is converted to the acylfulvene (R₁=H, R₂ =H, R₃ =CH₃) in 55% yield. A large number of analogs may beobtained from the acylfulvene by modifying the R₂ substituent, e.g., R₂=hydroxymethyl, bromo, iodo, chloro, fluoro, nitro, p-hydroxybenzyl,p-methoxybenzyl. R₂ can also be polynuclear or heterocyclic aromaticgroups. ##STR10##

The R₁ group may be acyl or alkyl. Analogs with different R₃ groups (andalso R₂ groups) can be prepared by total synthesis as outlined.##STR11##

Aldol condensation of 1,1-diacetylcyclopropane with the dianion derivedfrom an appropriately substituted cyclopentadiene gives an intermediatewhich is readily converted to the acylfulvene. A wide variety ofcompounds are possible since R₂ and R₃ may be alkyl, aryl or substitutedalkyl or aryl groups.

Synthesis of acylfulvene. Illudin S 2 g (9.2 mmol) was dissolved in 700mL water followed by addition of 4 M H₂ SO₄ (236 mL). The solution wasstirred overnight, and extracted with ethyl acetate. The organic phasewas washed with saturated NaHCO₃, water and brine, dried over MgSO₄ andconcentrated. Chromatography on silica with hexane and ethyl acetateafforded 0.82 g acylfulvene (50%). ¹ H NMR δ0.73 to 1.50 (m, 4 H), 1.38(s, 3 H), 2.00 (s, 3 H), 2.15 (s, 3 H), 3.93 (s, 1H), 6.43 (s, 1 H),7.16 (s, 1 H); MS m/Z 216 (M+), 202 (M+-CH₂), 188 (M+-CH₂ CH₂), 173(M+-2CH₂ CH₃), 170 (M+-2CH₂ -H₂ O). UV 325 nm (8.3×10³), 235 nm(16.6×10³). The compound has the structure: ##STR12##

Synthesis of 6-hydroxymethylfulvene. Acylfulvene (550 mg, 2.5 mmol) wasdissolved in 40 mL THF and 30% formaldehyde-water solution (40 mL) wasadded. 4 N H₂ SO₄ solution (26.4 ml) was added to bring the finalconcentration of H₂ SO₄ to 1 N The solution was stirred overnight andextracted with ethyl acetate. The organic phase was washed withsaturated NaHCO₃ solution, water and brine and dried over MgSO₄.Chromatography on silica gel with hexane and ethyl acetate gave 400 mghydroxymethylfulvene (64%). ¹ H NMR δ0.72 to 1.48 (m, 4 H), 1.38 (s, 3H), 2.15 (s, 3 H), 2.19 (s, 3 H), 3.90 (s, 1 H), 4.66 (d, J=2.1 Hz, 2H), 7.10 (s, 1 H). MS m/Z 246 (M+), 228 (M+-H₂ O), 218 (M+-CH₂ CH₂), 186(M+-CH₃ -CH₂ -CH₂ -OH), 185 (M+-H₂ O-CH₂ -CH₂ -CH₃). UV 233 nm(1.0×10⁴), 325 nm (7.7×10³). The compound has the structure: ##STR13##

Iodofulvene. To a solution of acylfulvene (60 mg, 0.28 mmol) in 15 mlCH₂ Cl₂ was added silver trifloroacetate (63 mg, 0.29 mmol). A solutionof iodine (70.5 mg, 0.28 mmol) in 8 mL CH₂ Cl₂ was added dropwise at 0°C. The mixture was stirred at that temperature for 3 hours then filteredthrough celite and eluted with ether. Concentration of the filtrate gaveiodofulvene as a red gum (73 mg, 77%). ¹ H NMR δ0.76 to 1.54 (m, 4 H),1.38 (s, 3 H), 2.14 (s, 3 H), 2.36 (s, 3 H), 3.87 (s, 1 H), 7.16 (s, 1H). MS m/Z 342 (M+), 314 (M+-CH₂ CH₂), 299 (M+-CH₂ CH₂ -CH₃), 29 6(M+-CH₂ CH₂ -H₂ O), 215 (M+-I), 187 (M+-I-CH₂ CH₂), 127 (I+). Thecompound has the structure: ##STR14##

Bromofulvene. Acylfulvene (60 mg, 0.28 mmol) was dissolved in 9 mlacetonitrile at 0° C. N-bromosuccinimide (50 mg, 0.28 mmol) was addedand the mixture was stirred at that temperature for 3.5 hours. Water wasused to quench the reaction and ether to extract the product. The etherlayer was washed with water and brine and dried over MgSO₄.Chromatography gave bromofulvene as orange crystals (77 mg, 94%;recrystalized from ether acetate-hexane, m.p.. 92°-94° C.). ¹ H NMRδ0.75 to 1.55 (m, 4 H), 1.40 (s, 3 H), 2.12 (s, 3 H), 2.33 (s, 3 H),3.89 (s, 1 H), 7.15 (s, 1 H). MS m/Z 295 (M+), 293 (M+-2), 267, 265,(M+-CH₂ CH₂), 252, 250 (M+-CH₂ CH₂ -CH₃), 249, 247 (M+-CH₂ CH₂ -H₂ O), 215 (fulvene-1). The compound has the structure: ##STR15##

p-Hydroxybenzylfulvene. Phenol (40 mg, 0.4 mmol) was added to a solutionof hydroxymethyl-fulvene (70 mg, 0.28 mmol) in dry CH₂ Cl₂ (25 mL). Themixture was cooled to -78° C. and boron triflouride etherate (0.3 mL,2.7 mmol) was added dropwise.. The reaction was stirred at thattemperature for 1 hour and water was added to quench the reaction. Theorganic layer was washed with H₂ O, NaHCO₃ and brine, and dried overMgSO₄. Chromatography on silica gel with hexane-ethyl acetate yielded 90mg (98%) of red crystals (m.p. 143°-144° C). ¹ H NMR δ0.59-1.43 (m, 4H), 1.36 (s, 3 H), 1.76 (s, 3 H), 2.07 (s, 3 H), 3.95 (s, 1 H), 3.97 (d,J=7.2 Hz, 2 H), 4.83 (s, 1 H), 6.74 (d, J=8.4 Hz, 2 H), 6.91 (d, J=8.4Hz, 2 H), 7.22 (s, 1 H). MS m/Z 322 (M+), 294 (M+-2 CH₂), 279 (M+-2 CH₂-CH₃), 251 (M+-2 CH₂ -CH₃ CO), 215, 107 UV 228 nm (12×10⁴, withinflections at 243 and 262 nm), 325 (7.1×10³), 410 nm (2.6×10³). Thecompound has the structure: ##STR16##

p-Methoxybenzylfulvene. Anisole (0.04 mL, 0.37 mmol) was added to asolution of hydroxymethylfulvene (10 mg, 0.04 mmol) in dry CH₂ Cl₂ (5mL). The mixture was cooled to -78° C. and boron triflouride etherate(0.04 mL, 0.36 mmol) was added dropwise. The reaction was stirred atthat temperature for 1 hour and water was added to quench the reaction.The organic layer was washed with H₂ O, saturated NaHCO₃ and brine, anddried over MgSO₄. Concentration of the solution gave a residue which wasdried in vacuo, yielding the product in quantitative yield (14 mg). ¹ HNMR δ0.59-1.40 (m, 4 H). 1.36 (s, 3 H), 1.76 (s, 3 H), 2.07 (s, 3 H),3.78 (s, 3 H), 3.95 (s, 1 H), 3.99 (d, J=16.12 Hz, 2 H), 6.81 (d, J-8.8Hz, 2 H), 6.96 (d, J=8.3 Hz, 2 H), 7.22 (s, 1 H). MS m/Z 336 (M+), 308(M+-CH₂ CH₂), 215, 121 ((CH₂ -Ph-OCH₃)+). UV 410 nm (2.7×10³), 325 nm(7.0×10³), 267 nm (1.0×10⁴), 245 nm (inflection), 226 nm (1.9×10⁴), 203nm (1.4×10⁴). The compound has the structure: ##STR17##

EXAMPLE XII In Vitro Cell Culture Studies of Acylfulvene Analogs

In vitro testing using cell culture assays demonstrated the6-hydroxymethylacylfulvene, bromoacylfulvene, and iodoacylfulveneanalogs were markedly toxic to the target tumor cells HL60 and MV522 atboth 2 and 48 hour exposure periods (Table 8 and Table 9). The relativetoxicity ratio (2 to 48 hour toxicity) suggested these analogs would bemore efficacious in vivo than either the parent Illudin S compound orthe analogs described in the original patent application.

                  TABLE 8                                                         ______________________________________                                        2 hour cytotoxicity of new analogs (as determined by                          inhibition of thymidine incorporation compared to                             original analogs and Illudin S                                                        2 hour IC50 values (nanomoles/liter)                                          HL60 Cells                                                                             8392 Cells   MV522                                           ______________________________________                                        Illudin S 10 ± 1  236 ± 22  19 ± 6                                   Dehydroilludin                                                                          377 ± 81                                                                              61,335 ± 9,810                                                                          1,826 ± 378                              Acylfulvene                                                                              998 ± 244                                                                             66,435 ± 13,006                                                                         727 ± 180                               6-hydroxy-                                                                              150 ± 11                                                                               7,359 ± 2,096                                                                          114 ± 28                                 methyl-                                                                       acylfulvene                                                                   acetate analog                                                                          3,333 ± 192                                                                           47,455 ± 2,951                                                                          1,066 ± 87                               of 6-HMAF                                                                     bromo-    803 ± 88                                                                              17,175 ± 890                                                                            4,180 ± 424                              acylfulvene                                                                   iodoacylfulvene                                                                         2,602 ± 345                                                                           10,331 ± 497                                                                             956 ± 152                               p-hydroxy-                                                                              264 ± 38                                                                               95,236 ± 11,984                                                                        1,180 ± 180                              benzylfulvene                                                                 p-methoxy-                                                                              1,964 ± 84                                                                            35,714 ± 7,292                                                                          2,045 ± 208                              benzylfulvene                                                                 ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        48 hour cytotoxicity of new analogs compared to original                      analogs and the parent Illudin S compound.                                             48 hour IC50 values (nanomoles/liter)                                         HL60 Cells                                                                             8392 Cells  MV522                                           ______________________________________                                        Illudin S   3 ± 1   8 ± 2    4 ± 1                                   Dehydroilludin M                                                                         296 ± 66                                                                               269 ± 100                                                                             313 ± 23                                 Acylfulvene                                                                              364 ± 74                                                                               833 ± 152                                                                             349 ± 23                                 6-hydroxymethyl-                                                                          4 ± 1  76 ± 4   73 ± 8                                   acylfulvene                                                                   acetate analog of                                                                        806 ± 30                                                                              4,434 ± 163                                                                            486 ± 42                                 6-HMAF                                                                        bromoacylfulvene                                                                         412 ± 21                                                                              1,186 ± 138                                                                            356 ± 61                                 iodoacylfulvene                                                                          290 ± 12                                                                              1,696 ± 183                                                                            556 ± 47                                 p-hydroxybenzyl-                                                                         382 ± 39                                                                              11,078 ± 388                                                                           615 ± 56                                 fulvene                                                                       p-methoxybenzyl-                                                                         1,051 ± 104                                                                           7,143 ± 244                                                                            1,548 ± 214                              fulvene                                                                       ______________________________________                                    

EXAMPLE XIII In Vivo Studies

Based on these in vitro screening results, one of the analogs, the6-hydroxymethylacylfulvene, was chosen for in vivo studies to determineif the analog was indeed more potent than the acylfulvene. The xenograftwas again the human lung adenocarcinoma MV522 as it is a nonresponsivemodel to conventional anti-cancer agents and kills by metastasis (notlocal tumor invasion). The conventional anti-cancer agents cis-platinum,taxol, mitomycin C, adriamycin, as well as Illudin S were chosen aspharmaceutical controls. A control group was included that received onlythe solvent used to dissolve the drugs, a 40% dimethylsulfoxide/normalsaline mixture (40% DMSO/NS). The 6-hydroxymethylacylfulvene analogactually induced tumor regression in animals. Actual regression oftumors by an anticancer agent has never been noted before in this model.There was no inhibition of tumor growth by conventional anticanceragents or by Illudin S (FIG. 10). Animals received only 9 doses of the6-hydroxymethylacylfulvene analog. There was no evidence of toxic sideeffects in these animals as evidenced by a decrease in activity, weightgain, food intake, or water intake. There was no significant increase inthe life span of cisplatinum, taxol, mitomycin C, and Adriamycin treatedanimals as compared control (DMSO/NS) treated animals. The Illudin Sactually caused premature death (drug toxicity) (Table 10). The6-hydroxymethylacylfulvene treated animals lived significantly longerthan controls (DMSO/NS treated), cisplatinum, taxol, mitomycin C, andAdriamycin treated animals (p<0.001 for all groups) (Table 10).

                  TABLE 10                                                        ______________________________________                                        Efficacy of 6-hydroxymethylacylfulvene analog,                                versus other agents, in the human lung adenocarcinoma                         MV522 metastatic lung tumor model - First experiment                          drug                   life span                                              ______________________________________                                        controls DMSO/NS IP 3×/WK                                                                      100 ± 7%                                            cis-platinum                                                                              3.2 mg/kg IP 1×/WK                                                                     102 ± 8%                                        taxol       4.0 mg/kg IP 5×/WK                                                                      100 ± 10%                                      mitomycin C 2.4 mg/kg IP 1×/WK                                                                     111 ± 2%                                        Adriamycin  2.6 mg/kg IP 1×/WK                                                                      98 ± 12%                                       Illudin S   2.5 mg/kg IP 3×/WK                                                                     <26%                                               [5/5 dead at only 3 doses]                                                    6-hydroxy-   10 mg/kg IP 3×/WK                                                                      233 ± 18%                                      methyl-                                                                       acylfulvene                                                                   ______________________________________                                    

The experiment was repeated with different dosages of the6-hydroxymethylacylfulvene analog to determine if a dose-responsepattern was present. Taxol and low dose Illudin S were again included aspharmaceutical controls (Table 11). A control group receiving only thepharmaceutical solvent (40% DMSO/NS) was again included. Tumorregression was again noted at 10 mg/kg, and inhibition of tumor growthwas noted with 1 and 5 mg/kg treatment. The taxol and low dose Illudin Sagain failed to inhibit tumor growth (FIG. 11). In this secondexperiment there was again no significant increase in the life span oftaxol or Illudin S treated animals as compared controls (40% DMSO/NStreated animals). The 10 mg/kg 6-hydroxymethylacylfulvene and 5 mg/kg6-hydroxymethylacylfulvene treated animals lived significantly longerthan controls (DMSO/NS treated), taxol, or Illudin S treated animals.The probability (or significance) value for 10 mg/kg6-hydroxymethylacylfulvene treated animals versus the controls, taxoltreated, and Illudin S treated animals was less than 0.001 in each case(p<0.001). The probability value for the 5 mg/kg6-hydroxymethylacylfulvene treated animals versus the controls, taxoltreated, and Illudin S treated animals was also less than 0.001 in eachcase (p<0.001). The 1 mg/kg 6-hydroxymethylacylfulvene treated animalsalso lived significantly longer than the control group (p<0.01).

                  TABLE 11                                                        ______________________________________                                        Efficacy of 6-hydroxymethylacylfulvene analog,                                versus other agents, in the human lung adenocarcinoma                         MV522 metastatic lung tumor model - Second experiment                         drug                   life span                                              ______________________________________                                        controls DMSO/NS IP 3×/WK                                                                      100 ± 16%                                                                  (100% by definition)                                   taxol       4.0 mg/kg IP 5×/WK                                                                     120 ± 10%                                                                  (not significant)                                  Illudin S  0.25 mg/kg IP 3×/WK                                                                     104% ± 20%                                      6-hydroxymethyl-                                                                           10 mg/kg IP 3×/WK                                                                     232 ± 20%                                       acylfulvene                                                                   6-hydroxymethyl-                                                                           5 mg/kg IP 3×/WK                                                                      154 ± 13%                                       acylfulvene                                                                   6-hydroxymethyl-                                                                           1 mg/kg IP 3×/WK                                                                      135 ± 10%                                       acylfulvene                                                                   ______________________________________                                    

The experiment was repeated for a third time (Table 12). The amount oftaxol administered IP was increased to demonstrate maximum dosage, and asubcutaneous dosage was added due to reports that this route may be moreefficacious. Adriamycin and mitomycin C were again included. Two of thenew acylfulvene derivatives (iodoacylfulvene and thep-hydroxybenzylfulvene) were included in this experiment. The diketoneanalog was included to demonstrate the marked improvement of the6-hydroxymethylacylfulvene. The 6-hydroxymethylacylfulvene markedlyincreased the life span versus controls and all other drug treatedanimals. The probability (or significance) value for the6-hydroxymethylacylfulvene versus all the control group, the mitomycin Ctreated group, the adriamycin treated group, and both of the taxoltreated groups was less than 0.0005 (p<0.0005). This is an extremelysignificant effect. It is important to note that based on the longevityof 2 particular animals, there is the possibility they may have beencured. Although the diketone also markedly increased the lifespan versuscontrols (p<0,002) and other drug treated animals, it was not aseffective as the 6-hydroxymethylacylfulvene. Note that the lifespan ofthe high dose taxol treated animals (6 mg/kg IP) not only decreasedbelow that of the 4 mg/kg taxol treated animals in the previousexperiment, but it was now less than the life span of untreated orcontrol animals. This indicates that the maximum dose for taxol had beenreached and drug toxicity was now killing the animals. The other newanalogs, the iodoacylfulvene and the p-hydroxybenzylfulvene are alsoeffective in this model.

                  TABLE 12                                                        ______________________________________                                        Efficacy of 6-hydroxymethylacylfulvene analog,                                versus other agents, in the human lung adenocarcinoma                         MV522 metastatic lung tumor model - Third experiment                          drug                           life span                                      ______________________________________                                        controls DMSO/NS IP 3×/WK                                                                          100 ± 29%                                       taxol          6.0 mg/kg IP 5×/WK                                                                       93 ± 22%                                   taxol         20.0 mg/kg IP 5×/WK                                                                      113 ± 22%                                   mitomycin C    2.4 mg/kg IP 1×/WK                                                                      149 ± 12%                                   adriamycin     2.6 mg/kg IP 1×/WK                                                                      105 ± 25%                                   diketone   30 mg/kg IP 3×/WK                                                          163 ± 6%                                                     (listed original application)                                                 iodoacylfulvene                                                                               20 mg/kg IP 3×/WK                                                                      120 ± 34%                                   p-hydroxybenzylfulvene                                                                        15 mg/kg IP 3×/WK                                                                      125 ± 16%                                   p-hydroxybenzylfulvene                                                                        20 mg/kg IP 3×/WK                                                                      126 ± 22%                                   6-hydroxymethyl-                                                                              10 mg/kg IP 3×/WK                                                                      >204%                                          acylfulvene   (2 animals alive, ? cured)                                      ______________________________________                                    

Although the invention has been described with reference to thepresently-preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

We claim:
 1. A therapeutic method of inhibiting rumor cell growth in asubject in need of said therapy comprising parenterally administering atherapeutic amount of a compound in a pharmaceutically acceptablecarrier, wherein said compound has the structure: ##STR18## wherein R isselected from the group consisting off ##STR19## and wherein said rumorcell is sensitive to inhibition by said compound and is selected fromthe group consisting of a myeloid leukemia cell, a T-cell leukemia cell,a lung carcinoma cell, an ovarian carcinoma cell, and a breast carcinomacell.
 2. The method of claim 1 wherein administering the compoundcomprises intravenously administering the compound to the subject. 3.The method of claim 2 wherein the therapeutic amount is between 30 to112,000 μg per kg of body weight.
 4. The method of claim 1 whereinadministering the compound comprises intraperitoneally administering thecompound to the subject.
 5. The method of claim 4 wherein thetherapeutic amount is between 300 to 112,000 μg per kg of body weight.6. The method of claim 1 wherein R is CH₂ OH.
 7. The method of claim 1wherein R is Br.
 8. The method of claim 1 wherein R is I.
 9. The methodof claim 1 wherein R is ##STR20##
 10. The method of claim 1 wherein R is##STR21##
 11. The method of claim 1 wherein R ##STR22##
 12. Atherapeutic composition comprising 30 to 100 mg of the compound of claim1 and a pharmaceutically acceptable carrier.